Lanes 1-7 : Anti-FH/Fumarase antibody [EPR21105] (ab233393) at 1/5000 dilution
Lanes 8-9 : Anti-FH/Fumarase antibody [EPR21105] (ab233393) at 1/1000 dilution

Lane 1 : HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg
Lane 2 : HEK-293 (human epithelial cell line from embryonic kidney) whole cell lysate at 20 µg
Lane 3 : Jurkat (human T cell leukemia cell line from peripheral blood) whole cell lysate at 20 µg
Lane 4 : NIH/3T3 (mouse embryo fibroblast cell line) whole cell lysate at 20 µg
Lane 5 : PC-12 (rat adrenal gland pheochromocytoma cell line) whole cell lysate at 20 µg
Lane 6 : Rat brain tissue lysate at 10 µg
Lane 7 : Rat spleen tissue lysate at 10 µg
Lane 8 : Human fetal brain tissue lysate at 10 µg
Lane 9 : Human fetal spleen tissue lysate at 10 µg

Secondary
Lanes 1-7 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Lanes 8-9 : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/4000 dilution

Developed using the ECL technique.

Predicted band size: 54 kDa
Observed band size: 48 kDa why is the actual band size different from the predicted?

Exposure times:

Lanes 1-5: 6 seconds.
Lanes 6 & 7: 8 seconds.
Lanes 8 & 9: 3 seconds.

Blocking and dilution buffer: 5% NFDM/TBST.

The lower mass bands could represent isoforms which have been described in the literature (PMID: 434773)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (human epithelial cell line from cervix adenocarcinoma) cell line labeling FH/Fumarase with ab233393 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining in HeLa cell line is observed. The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) at 1/200 dilution (red).

Secondary antibody only control: Used PBS instead of primary antibody, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

Immunohistochemical analysis of paraffin-embedded rat kidney tissue labeling FH/Fumarase with ab233393 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Granular, cytoplasmic and nuclear staining on rat kidney (PMID: 17111171) is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Immunohistochemical analysis of paraffin-embedded mouse kidney tissue labeling FH/Fumarase with ab233393 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Intensive granular and weak nuclear staining in mouse kidney is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

FH/Fumarase was immunoprecipitated from 0.35 mg of HEK-293 (human epithelial cell line from embryonic kidney) whole cell lysate with ab233393 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab233393 at 1/2000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution.

Lane 1: HEK-293 whole cell lysate 10 μg (Input).
Lane 2: ab233393 IP in HEK-293 whole cell lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab233393 in HEK-293 whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 10 seconds.

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized HeLa (human epithelial cell line from cervix adenocarcinoma) cell line labeling FH/Fumarase with ab233393 at 1/500 dilution (red) compared with an Isotype control details (ab172730) (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077), at 1/2000 dilution was used as the secondary antibody.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (mouse embryo fibroblast cell line) cells labeling FH/Fumarase with ab233393 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining in NIH/3T3 cell line is observed. The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) at 1/200 dilution (red).

Secondary antibody only control: Used PBS instead of primary antibody, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

Immunohistochemical analysis of paraffin-embedded human kidney tissue (left) and human clear cell renal cancer tissue (right) tissue labeling FH/Fumarase with ab233393 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Intense granular staining on human kidney whereas weaker staining in clear cell renal cancer tissue (PMID: 21695080) is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).